Abstract Tumor necrosis factor (TNF) ligands and TNF receptors (TNFRs) are essential regulators of the immune response. Dysregulation of TNF plays a role in the pathology of many autoimmune diseases that currently afflict more than 23.5 million people in the United States. Therapeutic targeting of TNFR1 signaling (e.g. for rheumatoid arthritis, and inflammatory bowel disease) is a billion-dollar industry. However, the available anti-TNF agents cause severe and adverse side effects. Thus, there is a desperate need to develop 'anti-TNFR' instead of 'anti- TNF' treatments in chronic inflammatory and autoimmune disorders. Despite some recent progress in this regard, state-of-the-art small molecule approaches have failed to uncover any high affinity small molecule inhibitors. In an attempt to jumpstart renewed and needed therapeutic discovery efforts, we have been building on existing yeast display/directed evolution technology to engineer high affinity TNFR ligands, in place of small molecules. Protein ligand scaffolds, peptides with high affinity and large surface area, are engineered by modulating amino acids in a select region, known as the paratope, of a protein while conserving a stable underlying scaffold. One particular example, the affibody domain, which has been extensively studied and improved by co-PI Hackel, has been effectively used as a ligand scaffold to numerous targets, with affinities as strong as 20 pM, and application to diagnostics, molecular imaging, and therapy. However, as we progressed towards high affinity binders to the TNFR family, we reached a familiar bottleneck in the field: how to direct the evolution of binders based not on affinity, but on functionality. While numerous platforms exist for discovery and evolution of protein binding, no robust methods have been established for the selection of precise biological activity (aside from general survival screens). Thus, the objective of this proposal is the development of a new technology for activity-based, high-throughput screening of protein ligands. In so-doing, we will discover novel, high-affinity inhibitors of TNFRs. Aim 1 will achieve discovery and evolution of a broad panel of strong binders to TNFRs, though the frequency of functional inhibitors is expected to be quite low. Aim 2 develops a technology to dramatically enhance the discovery of functional binders, which will have broad utility for all active ligand screening in addition to a focused benefit on the current TNFR antagonist development.